Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.


Introduction
Large numbers of protein-based therapeutics that could potentially enhance tissue regeneration have been identi ed, such as growth factors, but their therapeutic value in clinical medicine has been limited due to the di culty of maintaining bioactivity of locally applied proteins in the protease-rich environment of regenerating tissues 1 .Although human diseases are often treated with systemically administered drugs, the systemic use of powerful biological agents has largely been ruled out due to safety concerns.Thus, pharmaceutical efforts aimed at enhancing tissue repair have been based on local application at the site of the injury 2 .
To facilitate systemic delivery in regenerative medicine, we previously sought to create a platform for systemic administration and target-speci c delivery that is based on wound-homing peptides.The peptides were identi ed through screening of phage libraries in vivo for peptides that home to skin wounds and transected tendon injuries 3 .A 9-amino acid cyclic peptide, CAR (sequence CARSKNKDC) was particularly effective in homing to wounds and shown to recognise angiogenic blood vessels in regenerating tissues 3 .Subsequently, CAR peptide was used to deliver a recombinant fusion protein, consisting of the anti-brotic protein decorin, into healing wounds 4 .CAR-mediated targeting enhanced the accumulation and anti-brotic activity of decorin in the wounds; promoting wound healing and suppressing scar formation 4 .
Here, we have investigated the effect of CAR peptide on wound healing and show that CAR alone, in the absence of a coupled therapeutic partner, has an inherent ability to promote wound healing.This observation led us to investigate how CAR accelerates wound repair.We found that CAR has little effect on granulation tissue formation but promotes re-epithelialisation of wounds and epithelial cell migration.

CAR peptide accelerates skin wound re-epithelialisation and closure
Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialise the epidermis 12,13 .CAR peptide is capable of targeting angiogenic vasculature in injured and in amed tissues enabling delivery to sites of injury 3,14−16 .When CAR-decorin wound healing studies were conducted 4 , preliminary indications of accelerated wound re-epithelialisation in the animals treated with CAR peptide alone (a control group to treatment with recombinant CAR-decorin fusion protein) were noted, even at a very low dose of CAR peptide (1 µg/kg daily) 4 .Therefore, we examined the potential therapeutic effect of systemic CAR peptide administration on skin wound healing.Systemic i.v.administration of CAR or mutant CAR peptides (mCAR; CARSKNKDC mutated to CAQSNNKDC in mCAR, which abolishes the homing activity almost completely) 3,4 , or BSA in PBS (Control) via tail vein injections was initiated 24 hours post-wounding.The treatment was continued for 5, 7 or 10 days in three independent treatment experiments with 24 mice in each treatment group.Based on previous CAR peptide-studies [14][15][16] , a treatment regimen of two daily doses of 3.0 mg/kg CAR peptide was selected.
Daily analysis of the wounds demonstrated that wounds treated with CAR peptide closed signi cantly faster than the control groups during the treatment trials (Fig. 1A/B).The wounds were signi cantly smaller in CAR-treated animals than in control groups from day 4 onwards (P < 0.05 CAR vs control; P < 0.001 CAR vs mCAR), exhibiting substantial decreases in open wound area from day 7 to 10 (Fig. 1A/B).Moreover, the percentage of wounds that showed complete closure was signi cantly higher in CAR peptide-treated animals than in control groups (Fig. 1C/D).
Having established a clear effect on macroscopic wound closure, histological analyses were carried out to determine the impact of CAR peptide on wound re-epithelialisation, contraction and granulation tissue formation.Excision wounds close via co-ordination of re-epithelialsation and wound contraction (approximately 60% and 40% contribution, respectively, in BALB/c mice) and re-epithelialisation and contraction can be reliably quanti ed 17 (Fig. 2A).Histological analysis of wounds on days 5, 7 and 10 showed that re-epithelialisation was signi cantly more advanced in the group that received CAR peptide than in the control groups.The size of the gap between epidermal tongues (WG X ), i.e. the region of wound remaining without epidermis, was 60% and 83% shorter in CAR-treated mice than in control groups 5 and 7 days after wounding, respectively (Fig. 2B/G).All CAR peptide-treated wounds had completed reepithelialisation by day 10, whereas part of the wounds remained without new epidermis in the control and mCAR-treated wounds (Fig. 2B/G).
As a large number of CAR-treated wounds had obtained complete re-epithelialization by days 5 and 7, the proportion of wounds with complete re-epithelialisation was determined.The percentage of wounds that had complete re-epithelialisation was signi cantly higher in the CAR-treated group than in control groups at all time-points examined (Fig. 2C).As CAR peptide treatment accelerated complete re-epithelialisation (days 7 and 10), the area of hyperproliferative epidermal tongues was assessed at the earliest time point (day 5).This area was signi cantly higher in CAR peptide-treated wounds than in other wounds (Fig. 2D).
After re-epithelialisation has taken place, wounds contract in size.Thus, we measured wound contraction next.While the widths of the wounds (W X ) were identical in all treatment groups on days 5 and 7, they were signi cantly smaller in CAR peptide-treated mice on day 10 (Fig. 2E).Together these results demonstrate that CAR-induced wound closure and re-epithelialisation were not caused by panniculus carnosus-driven wound contraction and demonstrate accelerated maturation of the wound following early completion of re-epithelialisation in CAR-treated wounds.While granulation tissue looked more mature in CAR peptide-treated animals at early time-points (days 5 and 7), as suggested by reduced brin clot, no difference in granulation tissue area was detected between the groups at any studied time-point (Fig. 2F/G).Again, these data are consistent with CAR peptide not inducing wound contraction.Thus, CAR peptide accelerates wound healing and maturation primarily by selectively promoting reepithelialisation.

CAR peptide promotes epithelial cell migration
To elucidate the mechanism by which CAR peptide accelerates wound re-epithelialisation, we performed immunohistochemistry (IHC) analyses of skin wounds in mice treated with systemic i.v.injections of CAR, mCAR and BSA/PBS (Control) and collected the tissue on days 5, 7 and 10 post-wounding (Figs.S1; S2).Proliferation within the whole activated epidermis, including both the proliferation zone and the newly formed migrating epidermis was assessed.We detected no signi cant differences in the numbers of Ki67-positive proliferating cells in epidermis or granulation tissue between the treatment groups at any studied time-point during the healing period (Fig. S1A/B/F).Accumulation of macrophages in granulation tissue was also similar between the treatment groups (Fig. S1C/F).Next, we analysed wound vascularisation (angiogenesis): a slightly higher density of blood vessels was detected in early granulation tissue in CAR peptide-treated wounds on day 5, but this was minor and did not persist at later timepoints (Fig. S1D/F).To test the effect of CAR on myo broblast transformation and subsequent wound contraction, we determined the number of cells expressing high levels of α-smooth muscle actin (α-SMA), i.e. myo broblasts, in the granulation tissue (Fig. S1E/F).Interestingly, CAR peptide-treated wounds had substantially fewer myo broblasts than other treatment groups; indicating that enhanced wound closure in the CAR-treated group was not due to myo broblast-driven contraction.
Thus, CAR peptide enhances re-epithelialisation but does not enhance cell proliferation, macrophage accumulation or vascularisation in wounded skin tissue, and CAR-enhanced wound closure takes place without increased activation of myo broblasts.These data suggest that CAR peptide enhances wound healing by direct and selective stimulation of the wound epidermis.These results led us to hypothesise that CAR-mediated wound healing is driven by promotion of epidermal cell migration.Live-cell imaging of keratinocyte migration, using HaCaT cells in scratch wound assays, revealed that CAR peptide accelerated keratinocyte migration on bronectin.Over a 20-hour period, CAR peptide enhanced scratch wound closure approximately two-fold, relative to mCAR peptide or vehicle controls (Fig. 3A; Supplementary Video 1).The ability of cells to migrate across a substrate is in uenced by both the speed and directionality of migration 18,19 .Cell tracking analysis revealed that CAR peptide signi cantly increased the speed of cell migration (Fig. 3B) but had no effect on directional persistence (Fig. 3C).Interestingly, when we assessed cell migratory pro les, CAR-stimulated cells often exhibited an initial suppression of cell motility relative to controls, followed by a signi cant surge in migration while mCAR and vehicle treated controls slowed down (Fig. 3D/E; Supplementary Video 2).Together, these data suggest that CAR peptide accelerates wound healing in vivo by promoting keratinocyte migration.

SDC4 regulates CAR uptake and cytoskeletal reorganisation
We have previously shown that CAR peptide requires HSPGs for cell binding and penetrating activity 3,4 .
Cell migration relies critically on the co-ordination of cytoskeletal and adhesion dynamics.So, we next analysed the effect of CAR peptide on cytoskeletal and adhesion complex organisation.Cells were plated on the central cell-binding domain of bronectin (50K/Fn6-10), prior to stimulation with CAR, mCAR or vehicle control.This approach, which prevents matrix-engagement of SDC4, enables experimental separation and interrogation of integrin-and syndecan-dependent signals and functions, as described previously 9,11,18 .
Treatment of cells expressing endogenous or re-expressed wild-type SDC4 induced a distinctive cytoskeletal reorganisation within 30 minutes of CAR stimulation, characterised by loss of actin stress bres, formation of actin-rich membrane ru es, dissolution/disassembly of α5β1-dependent adhesion complexes and accumulation of α5β1 in intracellular vesicles (Fig. S3A/Bi).However, while a subset of cells continued to exhibit this morphology, following 120 minutes of CAR stimulation approximately 80% of SDC4-expressing cells underwent further cytoskeletal reorganisation and exhibited pronounced stress bres and elevated levels of α5β1 integrin at the cell-matrix interface (Fig. S3A/Bi).By contrast, mCAR treatment had no discernible effect on cytoskeletal architecture or integrin distribution, compared with the vehicle control.Importantly, Syn4-/-cells did not undergo cytoskeletal or adhesion complex reorganisation following CAR peptide treatment (Fig. S3Bi/Bii).Thus, stimulation of cells with CAR peptide induces cytoskeletal and α5β1 integrin adhesion complex remodelling in a SDC4-dependent manner.

SDC4 and bronectin are selectively expressed in migrating epidermis of skin wounds
As analyses in well-characterised cell models of SDC4-mediated functions suggested that SDC4 is required for CAR peptide internalisation, and that CAR regulates SDC4-dependent cytoskeletal dynamics, we assessed SDC4 in wounds.Fibronectin is the main endogenous SDC4 ECM ligand that activates SDC4-dependent pro-migratory pathways 5,9,11 and is expressed abundantly in skin wounds.Thus, we explored the expression and distribution of SDC4 and bronectin in skin wounds.In line with previous studies 20 , antibody staining revealed strong SDC4 expression in migrating epidermis (epidermal tongues), some expression in the dermis, mainly in blood vessels, but also in the granulation tissue (Fig. S6A/F).IHC double staining for SDC4 and bronectin showed that, where SDC4 was localised within migrating epidermis, the expression pattern of bronectin is the opposite; exhibiting no expression in migrating epidermis but abundant expression throughout underlying granulation tissue (Fig. S6B/C/Ci).While SDC4 was expressed throughout the epidermal tongues only cells directly contacting granulation tissue (basal keratinocytes) would have an opportunity to interact with bronectin.Double staining of SDC4 and keratinocyte-speci c marker cytokeratin 17 (CK17), con rmed the epithelial distribution of SDC4 and demonstrated co-expression in the migrating epidermis of skin wounds (Fig. S6D/E).
To further understand the expression of SDC4 during wound healing, we analysed single-cell transcriptomics data published on wound healing.We selected data from Haensel et al. 21for further interrogation because the study reports single-cell RNA sequencing (scRNA-Seq) data from normal and wounded mouse skin for all major skin cell populations (Fig. 5G) 21 .Our analysis of the scRNA-Seq skin wound dataset revealed very high SDC4 expression in all keratinocyte populations involved in wound healing, in contrast to substantially lower expression in different broblast populations (Fig. 5H/I).By contrast, expression of bronectin (FN1) is largely restricted to broblasts, especially those in an in ammatory state (Fig. 5H/I).Together, the IHC and scRNA-Seq analyses suggest that SDC4 is primarily expressed in the migrating epidermis in healing mouse wounds, whereas bronectin synthesis is restricted to underlying broblast populations.
As SDC4 is selectively expressed in wound epithelia, CAR promotes keratinocyte migration and CAR can use SDC4 to enter cells, ow cytometry was used to quantitatively determine the role of SDC4 in CAR peptide uptake in keratinocytes.Uptake of CAR-FAM peptide by HaCaT cells was signi cantly higher than mCAR-FAM and siRNA-mediated suppression of SDC4, with two different oligonucleotide sequences, reduced CAR-FAM uptake to basal levels (Fig. 5J).Thus, SDC4 is required for the uptake and internalisation of CAR peptide in epithelial cells.

CAR peptide regulates SDC4-dependent ARF6 activity
The vesicular distribution of CAR and SDC4 (Fig. 4B/Bi), suggested that their functional relationship may involve tra cking mechanisms.SDC4 is a direct regulator of receptor recycling and ligand-engagement of SDC4 modulates activation of the small GTPase ARF6 to spatially and temporally control integrin recycling and matrix-engagement 11,22 .ARF6 is activated upon ECM engagement and regulates recycling of receptors from intracellular vesicles to the plasma membrane 11,23 , and we have shown that SDC4 controls ARF6 activity to regulate differential recycling of integrins to co-ordinate cell migration 11,22 .
The fact that SDC4 is required for internalisation of CAR (Figs. 4A/B; 5J), co-localises with internalised CAR peptide (Fig. 4B) and regulates CAR-dependent modulation of integrin-mediated adhesion complexes (Fig. S3), and that CAR promotes epithelial cell migration (Fig. 3), prompted us to test whether CAR peptide regulates SDC4-dependent ARF6 activity in keratinocytes.Effector pull-down assays in HaCaT cells showed that stimulation with CAR peptide dynamically regulated ARF6 activity; inducing initial suppression of ARF6 activation, followed by a subsequent activation of ARF6 that was approximately 2.5-fold higher than in mCAR-treated cells (Fig. 6A/B).Likewise, CAR stimulation in HaCaT cells transfected with non-targeting control siRNA rapidly suppressed ARF6 activity by approximately 25%, followed by a substantial increase in ARF6 activity that was signi cantly higher than baseline and control conditions.However, siRNA-mediated knockdown of SDC4 abrogated the ability of CAR to regulate ARF6 activity (Fig. 6C/D/E).Thus, these time-course experiments demonstrate that CAR peptide dynamically controls ARF6 activation, resulting in ARF6 hyperactivity, and that SDC4 is essential for this effect.
Having demonstrated a link between CAR peptide and SDC4-mediated regulation of ARF6 activity, we analysed ARF6 expression in the scRNA-Seq skin wound healing dataset 21 .Interestingly, the highest levels of ARF6 expression were detected in differentiating and basal keratinocytes, (Fig. S4A/B); two cell types with a key role in wound re-epithelialisation 24 and high levels of SDC4 expression (Fig. 5H/J).Demonstrating that keratinocytes in mouse excisional skin wounds express high levels the key molecules associated with the CAR-stimulated signalling response.
We next assessed the impact of depleting ARF6 expression on CAR-mediated cytoskeletal and integrinmediated adhesion reorganisation.Intriguingly, under steady-state conditions on the integrin-binding central cell-binding domain of bronectin (50K/Fn6-10) and in the absence of peptide stimulation, siRNAmediated depletion of ARF6 resulted in loss of actin stress bres, formation of actin-rich membrane ru es, and small and/or indistinct α5β1-dependent adhesion complexes (Fig. S5A/B).These morphological changes are reminiscent of the CAR-induced cytoskeletal and integrin reorganisation elicited by short-term stimulation with CAR peptide (Fig. S3A/B); a treatment that initially suppresses ARF6 activity (Fig. 6).Moreover, ARF6 knockdown inhibited the stress bre and α5β1-dependent adhesion complex formation triggered by long-term CAR stimulation (Fig. S5A/B).Given that CAR stimulation induces an initial suppression of SDC4-dependent ARF6 activity followed by a substantial SDC4dependent increase in ARF6 activity, these data suggest that the effect of CAR peptide on cytoskeletal and adhesion dynamics is dependent on both ARF6 and SDC4.

CAR promotes SDC4-and ARF6-dependent keratinocyte migration
The preceding data demonstrated that CAR peptide, which promotes wound healing (Figs.2/3), is internalised with SDC4, induces SDC4-dependent ARF6 activation and triggers SDC4-and ARF6dependent cytoskeletal reorganisation (Figs.4; 6; S3; S5).As in vivo and cell biological data suggested that CAR induces wound healing by increasing epidermal cell migration (Figs.2; 3), we sought to determine whether SDC4 and ARF6 are required for CAR-dependent epidermal cell migration.
To analyse the contribution of SDC4 to CAR-stimulated epithelial cell migration, we assessed the effect of siRNA-mediated suppression of SDC4 expression on HaCaT motility on bronectin in scratch wound migration assays, following treatment with CAR, mCAR or vehicle control.SDC4 depletion, using two independent siRNA oligonucleotides, completely eliminated the increase in scratch wound closure and epithelial cell migration triggered by CAR peptide stimulation (Figs.7A-D; S6A; Supplementary Videos 3 &  4).As observed previously, in control cells CAR modulated migration speed, but had no effect on directionality.Likewise, SDC4 knockdown also did not affect directional persistence (Fig. S6B).As we previously observed in wild-type cells, in control siRNA cells CAR triggered an initial inhibition of migration followed by a surge in cell motility.Interestingly, both the initial CAR-induced suppression and subsequent increase in migration were dependent on SDC4 expression (Fig. 7B-D; Supplementary Videos 3 & 4).
As SDC4 is required for CAR-stimulated ARF6 activation and keratinocytes express high levels of ARF6 in wounds (Fig. S4), we next assessed cell migration following ARF6 knockdown.These experiments revealed that, while CAR peptide accelerates scratch wound closure and epithelial cell migration in nontargeting siRNA-expressing cells, depletion of ARF6 inhibited CAR-dependent migration (Figs.7E-H; S6C/D; Supplementary Videos 5 & 6).Thus, SDC4 regulates CAR-mediated ARF6 activity and both SDC4 and ARF6 are required for the pro-migratory effects of CAR peptide in keratinocytes.

CAR peptide requires syndecan-4 to accelerate wound reepithelialisation
Mechanistically, cell-based analyses suggested that SDC4 is required for internalisation of CAR peptide and that CAR stimulates SDC4-dependent ARF6 activity to control engagement of α5β1 integrin and epithelial cell migration.As our previous experiments suggested that CAR peptide accelerates wound healing in vivo by promoting keratinocyte migration (Figs.2; 3; 4; S1), we sought to determine whether SDC4 is required for CAR peptide to stimulate wound re-epithelialisation in vivo.Skin wounds in SDC4 knockout (KO) and wild-type mice were treated with CAR peptide.Systemic i.v.administration of two daily doses of 3.0 mg/kg CAR or BSA/PBS via tail vein injections was initiated 24 hours post-wounding.On day 7 post-wounding, skin wound tissues were collected and histologically analysed.As we have observed previously (Figs.2/3), CAR peptide enhanced wound re-epithelialisation of wild-type SDC4expressing mice.By contrast, wound re-epithelialisation was not accelerated in SDC4 KO mice (Fig. 8A/B/F).Thus, the size of the gap remaining without epidermis (WG X ) following CAR treatment was signi cantly smaller in SDC4 wild-type mice but was not in uenced in SDC4 KO mice (Fig. 8A).CAR peptide also increased the incidence of wounds with complete re-epithelialisation from 14-41% in SDC4 wild-type mice (Fig. 8B).Moreover, CAR increased the area of hyperproliferative epidermal tongues by 60% in normal mice but had no effect in SDC4 KO mice (Fig. 8C).As observed previously at 7 days postwounding (Fig. 3E/F), CAR treatment did not modulate overall wound width (W X ), ruling out contractiondriven wound closure, nor the area of granulation tissue relative to control treatment in either wild-type or SDC4 KO mice (Fig. 8D/E).
Finally, we examined homing of CAR peptide to SDC4 WT and KO wounds using FAM-labelled CAR peptide for the nal treatment dose.CAR peptide administered 4 hr before sacri ce was detected at high levels in SDC4 WT wounds, but not in SDC4 KO wounds (Fig S7).In SDC4 expressing mice, CAR peptide was recruited predominantly to the hyperproliferative epidermis, but could also be detected in vascular structures within granulation tissues and in the dermis; areas where SDC4 was also detected in wounds (Fig. 8G).Together these data demonstrate that CAR peptide utilises SDC4 to home to and penetrate wound tissue, but also uses SDC4-dependent signalling to promote wound healing and reepithelialisation.Thus, CAR appears to be an injury-targeting peptide, with potential utility for early intervention in wound healing.

Discussion
We show here that a homing peptide, named CAR, originally discovered as a wound-homing peptide and used as a delivery vehicle for therapeutics [2][3][4] , also possesses an inherent ability to accelerate wound healing.We further show that CAR peptide acts by activating SDC4-dependent mechanisms to drive epithelial cell migration.
The CAR peptide is an example of the propensity of in vivo phage display screening for homing peptides to yield peptides that are bioactive, in addition to being able to home to a particular target in the body.Earlier examples of such peptides include the tumour-penetrating peptides iRGD and LyP-1; iRGD has an inherent anti-metastatic activity 25 and LyP-1 elicits apoptosis in tumour cells and tumour macrophages 26,27 .The reason for this inherent bioactivity is that peptides interact with binding pockets in proteins, and those pockets are generally important active sites (for discussion see 28 ).CAR peptide is different from the existing bioactive peptides that bind to proteins in that its target molecule is a carbohydrate, the heparan sulfate glycosaminoglycan (GAG) chains of HSPGs 3,4 .CAR contains a classical heparin-binding domain (HBD) that has high homology with the HBD of bone morphogenetic protein-4, and we have previously shown that CAR binds to heparan sulfate and heparin 3,4 .HBD-GAG interactions are thought to be mostly charge mediated, i.e. dependent on attraction between basic residues in the HBD and sulfated sugar residues in the GAG 6,7,29,30 .HSPGs are ubiquitous, however the selective homing of CAR to angiogenic vessels and wound tissue suggests additional elements to the CAR speci city, such as a GAG sulfation pattern that creates a molecular signature characteristic of regenerating tissues.This scenario is supported by the recent demonstration that SDC4 expression is very low or absent from normal quiescent blood vessels, but it is the SDC-family member exhibits enhanced expression in angiogenesis 31 .Furthermore, it was also shown that owing to differences in the heparan sulfate chains of SDC2 and SDC4 (de ned by a unique protein sequence in SDC2 ectodomain), the heparin-binding growth factor, vascular endothelial growth factor-A (VEGFA), binds only to SDC2 and not to SDC4 32 .Thus, two factors, distinct GAG chains on speci c HSPGs and the selective overexpression of SDC4 in migrating epithelia and angiogenic vessels may contribute to the selectivity of CAR to wounds.
The targeted, organ-and cell-speci c mode of action of CAR makes it possible to use systemic administration of the peptide to accelerate wound healing, which circumvents limitations of local treatments, such as di culty in maintaining the activity of local agents in the wound environment.
Systemic treatment is also advantageous when the injured site cannot be accessed by topical application or multiple tissues are injured simultaneously.Our results only address the treatment of skin wounds, so it will now be important to determine whether the bene cial activities of CAR extend to other tissues.It is known that CAR homes to injuries in tendon 3 as well as to diseased tissue in pulmonary arterial hypertension, bronchopulmonary dysplasia, cancer (human tumour xenografts), aortic aneurysms, retinopathies, muscular dystrophies and myocardial infarction 14-16,33−38 .In line with CAR peptide homing to tissue injuries and these diseases, the upregulation of SDC4 expression has been described in all of these instances 7,20,31,36,39,40 .
Several lines of evidence from our study indicate that CAR promotes wound healing through selective engagement of SDC4.First, wounds in SDC4 knockout mice were impervious to the CAR-mediated effect.Second, our mechanistic data shows that SDC4 is required for internalisation of CAR peptide and that CAR modulates SDC4-dependent ARF6 activity to drive epithelial cell migration.Third, CAR peptide induced SDC4-and ARF6-dependent redistribution of α5β1 integrin, indicating that CAR regulates cell migration by orchestrating integrin engagement.Finally, SDC4 is expressed de novo on keratinocytes in the migrating epidermis (epidermal tongues), and re-epithelialisation is the main aspect of wound healing that is affected by CAR.
We also found that although the size of the granulation tissue was similar in the CAR-treated and control wounds, there was a striking difference in the number of myo broblasts in the two treatment groups; the CAR-treated wounds almost completely lacked myo broblasts, whereas myo broblasts were abundant in the control-treated wounds.Myo broblast-driven contraction of loose granulation tissue into a scar completes wound healing, but this process comes at a price, with formation of a permanent scar 41 .As there are fewer myo broblasts in CAR-treated wounds, CAR may also reduce scarring.This scenario is supported by recent ndings that SDC4 presence suppresses the development of brosis in brotic disease models [42][43][44] .However, this remains to be studied because the 10-day observation period used assess re-epithelialisation in this study was too short to assess permanent scar formation.
The SDC4-dependent cell migration pathway that underpins the mechanistic basis of CAR-accelerated wound healing, has been characterised in detail 9-11, 18,45 .While many ECM proteins contain HBDs, bronectin is the main endogenous SDC4 ECM ligand that activates these pro-migratory pathways 5,9 .Plasma bronectin is a major component of the blood clot that forms immediately after wounding and bronectin is also abundantly expressed by broblasts in granulation tissue 46 .Although plasma bronectin is not essential for wound healing 47 , cellular bronectin is 48 .Lack of bronectin extra domain A (EDA) leads to selective defects in wound re-epithelialization 48 .Thus, it is thought that bronectin provides a bed/scaffold for migrating epidermis to close the wound 48 .Yet our analysis of wound tissue indicates that very few migrating keratinocytes are in contact with bronectin during wound repair.Thus, it is probable that a large proportion of keratinocytes express unligated SDC4 that can receive an additional migration-promoting stimulus from CAR.Thus, we propose a model whereby, in the early stages following wounding, elevated SDC4 expression promotes CAR targeting to the site of injury, but also provides a reservoir of unengaged receptors that are available to be bound by the peptide to accelerate the endogenous wound healing response selectively on the epidermis.Mechanistically, our data indicate that ARF6 is the downstream effector of SDC4 signalling triggered by CAR to drive epithelial cell migration.Accumulating evidence suggests that integrin recycling plays a key role in cell migration 49,50 .As SDC4 regulates ARF6 to co-ordinate heterodimer-speci c recycling of integrins to the plasma membrane 11 it is likely that CAR peptide regulates cell migration and wound healing by driving recycling of integrins.Indeed, the SDC4-mediated activation of ARF6 induced by CAR, and the SDC4-and ARF6-dependent redistribution of α5β1 in response to CAR stimulation, are consistent with CAR regulating cell migration and wound healing by orchestrating α5β1 engagement.However, as SDC4 is also a growth factor co-receptor 5 , CAR-dependent ARF6 activation may also regulate growth factor receptor tra cking to co-ordinate cell migration and wound healing 22 .A signi cant level of crosstalk exists between adhesion receptors and growth factor receptors and this interplay has critical roles in cell migration 51,52 .Reciprocal, and mutually regulatory, tra cking mechanisms are one of the major mechanisms by which adhesion receptor and growth factor receptor signals are integrated 49,51,52 .
Thus, as syndecans and ARF6 regulate tra cking of both integrins and growth factor receptors 9,11,22 , it is possible that CAR may co-ordinate ligand engagement and signalling of multiple pro-migratory receptors.
Recent papers report the use of brin-conjugated peptides as a locally administered biomaterial to enhance wound healing 53 .The authors attributed the effects of these biomaterials on wound healing to growth factor retention afforded by the growth factor binding to the HBD of these peptides in the wounds 53 .Since these peptides contain HBD and bind to syndecans 53,54 , there is some similarity to our work.However, we use soluble peptide, that accumulates in the wounds by homing and cell penetration and is not covalently anchored to ECM, therefore it is unlikely that growth factor retention is a signi cant part of its mode of action.Moreover, our treatment is systemic which offers considerable advantages over local administration.Furthermore, these topically administered, syndecan-binding peptides are also functionally different from CAR in that their activities include, for example, stimulation of proteolytic activity by matrix metalloproteinases 55 .
Finally, the classical view of the role of heparan sulfate in the biology of heparin-binding growth factors is that their binding to HSPGs merely provides a way to concentrate and present growth factors to a signalling receptor, such as a receptor tyrosine kinase 29,30 .As the CAR peptide stimulates wound healing through SDC4-/HSPG-dependent signalling, it may be that heparin-binding growth factor signalling is more complex than currently thought, and that HSPGs, in addition to concentrating and presenting growth factors, also act as signalling receptors in concert with conventional growth factor receptors.
Defective wound healing has been reported in SDC4 knockout mice as well as in mice with a conditional, keratinocyte-speci c ARF6 deletion 6,7,56,57 .These studies, when considered alongside our data, highlight the key roles that both SDC4 and ARF6 play in wound healing and suggest that pharmaceutical manipulation of these pathways to promote wound healing may be therapeutically tractable.Thus, CAR peptide may provide a new way of enhancing wound healing by systemic treatment, and perhaps tissue regeneration in general.Indeed, it has been shown that SDC4 also plays key roles in skeletal muscle regeneration 58,59 , epithelial regeneration in experimental colitis models 60 and fracture and cardiac repair 36,61 .We have not seen any obvious toxicities in any of the many mice we have treated with CAR over extended periods of time.Moreover, CAR is active in human cells and tissues: the human keratinocyte cell line we used in this study responded to CAR with increased migration, and previous studies have shown that CAR binds to and penetrates human endothelial cells and homes to human tumour xenografts in vivo 14 .These features bode well for the translational prospects of CAR.

Mouse Husbandry and Derivation
Mice were fed with standard laboratory pellets and water ad libitum.All animal experiments were performed in accordance with protocols approved by the National Animal Ethics Committee of Finland, the institutional animal care and use committees of the Sanford Burnham Prebys Medical Discovery Institute (La Jolla, CA, USA) and the University of California at Santa Barbara (Santa Barbara, CA, USA).
In this study BALB/c mice and SDC4 KO mice have been used.The generation of SDC4 KO mice has been described in detail elsewhere 62 .SDC4 KO mice were obtained from Dr. Mark Bass (University of She eld, She eld, UK).Before initiating experiments, SDC4 KO mice were re-derived, backcrossed eight generations with C57BL/6 strain (Harlan) to obtain SDC4 expressing (wild-type, WT) and SDC4 KO strains in the same genetic background (littermates).Then homozygous SDC4 KO animals were bred.The genotype was determined in each animal by PCR.

Peptide Synthesis
CAR and modi ed CAR peptides (CAR: CARSKNKDC; mCAR: CAQSNNKDC) were synthesised with an automated peptide synthesiser by using standard solid phase uorenylmethoxycarbonyl chemistry.
During synthesis, some batches of peptides were labelled with uorescein amitide (FAM) using an aminohexanoic acid spacer as described previously for use in imaging studies 16 .The peptides were stored in -20°C, dissolved in PBS immediately before experiments, kept on ice, shielded from light and used fresh (within the same day).
Wound Healing Model and Treatment Schedule 8-10-week male mice (BALB/c, or SDC4 KO C57BL/6 and SDC4 WT C57BL/6 littermates), weighing 23-28 g, were used in wound studies.Mice were anaesthetised with 4% iso urane and 1.5 l/min of oxygen, and the anesthesia was maintained at ≈ 1.5% iso urane at 1 l/min of oxygen.Skin was shaved, cleaned, and disinfected with betadine and 70% alcohol.Treatment trials were conducted on mice that had circular, 6 mm diameter, full-thickness excision wounds (including panniculus carnosus muscle) in the dorsal skin 4 .The wounds were rst marked by a biopsy punch and then cut with scissors.All skin wounds were left uncovered without a dressing.
The treatments were started 24 hours after wounding and consisted of two daily tail vein injections.This systemic peptide injection schedule continued until the sacri ce of the animals.The dose for CAR and mCAR peptides was 3.0 mg/kg (approximately 62.5 µg per animal) per injection in 100 µl PBS on the basis of previous CAR peptide treatment studies [14][15][16] .BSA (62.5 µg) was used as a control protein in PBS injections.At the end of the treatment period, the animals were sacri ced, the wounded tissue collected and processed for further analyses.

Morphological Assessment of Wound Closure
After the surgery and daily after that until the sacri ce, the wounds were photographed digitally.Two 2 x 2 cm cardboard squares were placed on both sides of the animal to adjust the digital pictures taken from various distances in relation to wounds and the total area of a wound was measured and analysed from digital photographs using ImageJ software (NIH) by manually drawing the edges of each individual wound as described previously 4 .

Histology
Mice were euthanised and perfused by intra-cardial injection of 4% paraformaldehyde (PFA).Excision of a rectangular section of skin containing all wounds, as well as underlying skeletal muscle, was performed to ensure the uninterrupted wound 3,4,14 .The "whole-mounted" sections were immobilised on lter paper, immersed in 4% PFA for additional overnight xation, and washed with physiological saline.Thereafter, the wounds were bisected, dehydrated, and embedded in para n.Longitudinal sections (6 µm) from the middle of the wound were stained with hematoxylin/eosin (HE) and/or processed for immunohistochemistry.

Quantitative Analysis of Histology and Immunohistochemistry
Two HE-stained sections from the middle of each wound were quantitatively evaluated, and the average of the two values was used as one value for each wound in the analysis.From all wound data belonging to the same treatment and time-point group the average mean was plotted.When the wound heals, new epidermis grows from both sides of the wound edges and is termed hyperproliferative epithelial tongues.
The area of epithelial tongues as well the gap between the two epithelial tongues of a wound (a measure of re-epithelialisation) was measured as described by Chen et al. 17 .Schematic representation in Fig. 2A.WG X : Gap between the two epithelial tongues on day X post-wounding.E1 X + E2 X : Hyperproliferative epidermis (HPE) length was used to calculate HPE area per wound.W X : As a readout of wound contraction, the total size/width of the entire wound was determined on day X by the distance between the closest hair follicles on each side of the wound.Depending on the experimental setup for histological analysis, day X corresponded to day 5, 7 or 10 post-wounding.The area of granulation tissue was determined by drawing its edges using free-hand tool.Image analysis and quanti cation of histological and IHC parameters were performed using Spectrum digital pathology system using the Aperio ScanScope CS and XT systems (Aperio, Leica Biosystems) as described previously elsewhere 4,64 .Slides were viewed and analysed with the Aperio ImageScope software (Leica Biosystems).The regions of interests were recorded for each wound.All quanti ed histochemical analyses (Ki-67, CD31, F4/80, α-SMA) were performed according to the protocols used to establish these algorithms for each respective staining 4,64 .Analysis of Ki67-positive cells in the epidermis extended between 1st hair follicles, to ensure inclusion of the whole activated epidermis, including the proliferative zone.The same microscope settings and downstream image processing parameters were used for all samples within each individual staining group.All analyses were carried out by adhering to ARRIVE 2.0 guidelines 63 .

Cell Culture
The HaCaT spontaneously immortal keratinocyte cell line was maintained in Dulbecco's Modi ed Eagles Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS).Immortalised SDC4 WT and KO mouse embryonic broblasts (MEFs) 18 , were grown at the large T-antigen permissive temperature of 33˚C in DMEM supplemented with 10% FBS, 2mM L-glutamine, and 20U/ml IFNγ (Sigma-Aldrich).Syn4WT MEFs were generated by retroviral transduction of a HA-tagged wild-type human SDC4 construct into SDC4 KO MEFs, as described previously 11,18 .Large T-antigen immortalised human dermal broblasts (TIFs) were grown in DMEM supplemented with 15% FBS and 2mM L-glutamine at 37˚C.

Immuno uorescence: Peptide internalisation
Cells were plated on sterile coverslips in full growth medium for 24 hours, prior to treatment with 10mg/ml FAM-conjugated CAR peptides (CAR-FAM).Following 8 hours incubation, cells were xed with 4% (wt/vol) paraformaldehyde, permeabilised for 2 minutes at RT with 0.5% (wt/vol) TritonX-100 in PBS − and blocked (0.1% BSA/0.1% sodium azide in PBS − (0.1/0.1 buffer)).Cells were stained for actin with AlexaFluor-594-conjugated phalloidin (Life Technologies) and/or SDC4 with 5mg/ml anti-SDC4 primary antibody (3644; BioVision) and AlexaFluor-647-conjugated A niPure Donkey anti-Rabbit secondary antibody (Stratech).Cells were mounted in ProLong Gold (Life Technologies) and immuno uorescent images were acquired on an Olympus IX71 using DeltaVisionRT software, Olympus 60x/NA1.40Plan Apo objective and Coolsnap HQ camera.The same microscope settings were used to acquire all images within each experiment.The same ImageJ settings were applied for all conditions within a single experiment.

Immuno uorescence: CAR-Induced Cytoskeletal Reorganisation
To test the role of CAR stimulation on focal adhesion and cytoskeletal organisation, in the absence of syndecan-4 engagement, cycloheximide-treated cells were spread on sterile coverslips coated with 10mg/ml central cell-binding domain of bronectin (Fn6-10/50K) for 120 minutes, as described previously 9,11,18 , then stimulated with 10mg/ml CAR peptide, mCAR peptide or vehicle control for 30 or 120 minutes before xation.Cells were xed with 4% (wt/vol) paraformaldehyde, permeabilised for 2 minutes at RT with 0.5% (wt/vol) TritonX-100 in PBS − and blocked in 0.1/0.1 buffer.MEFs were stained for α5 integrin using 10mg/ml anti-mouse CD49e primary antibody (5H10-27(MFR5); BD Biosciences) and AlexaFluor-488-conjugated A niPure Donkey anti-Rat secondary antibody (Stratech) and stained for actin with AlexaFluor-594-conjugated phalloidin (Life Technologies).TIFs were stained for α5 integrin using 10mg/ml anti-human α5 integrin primary antibody (mab11; puri ed in-house from hybridoma) and AlexaFluor-488-conjugated A niPure Donkey anti-Rat secondary antibody (Stratech) and stained for actin with AlexaFluor-594-conjugated phalloidin (Life Technologies).Immuno uorescent images were acquired on an Olympus IX71 using DeltaVisionRT software, 60x/NA1.40Plan Apo or 40x/NA0.85Uplan Apo objectives and Coolsnap HQ camera.The same microscope settings were used to acquire all images within each experiment.The same ImageJ settings were applied for all conditions within a single experiment.

Flow Cytometry
To detect levels of cell surface SDC4 expression, cells were detached using enzyme-free Hanks'-based cell dissociation buffer (Life Technologies) and washed with 0.1/0.1 buffer.Cells were incubated with 10µg/mL primary antibody (5G9, anti-Human SDC4) for 30 minutes at 4°C in 0.1/0.1 buffer at 4°C, washed three times, and incubated with AlexaFluor 488-conjugated secondary antibody at 4°C for 30 minutes.Cells were analysed on an Attune NxT Flow Cytometer (ThermoFisher Scienti c) and analysed using FlowJo software.
Peptide Uptake Assay 24 hours post-transfection, HaCaT cells were plated on 10cm dishes in full growth medium for 24 hours, prior to treatment with 0.1mg/ml FAM-conjugated CAR peptide (CAR-FAM), 0.1mg/ml FAM-conjugated mCAR peptide (mCAR-FAM) or vehicle control (Nil).Following 4 hours incubation, cells were detached using enzyme-free Hanks'-based cell dissociation buffer (Life Technologies), to ensure retention of peptide associated with trypsin-cleavable cell surface HSPGs as well as internalised peptide.Cells were washed three times with 0.1/0.1 buffer, resuspended in PBS and analysed on an Attune NxT Flow Cytometer (ThermoFisher Scienti c) and analysed using FlowJo software.To normalise data between replicate experiments, mean uorescence intensity was normalised relative to the signal for mCAR-FAM in control knockdown cells.

Scratch Wound Cell Migration Analysis
Glass-bottom 24-well plates were coated with plasma bronectin from solution (10 µg/ml).HaCaT keratinocyte cells were plated at near con uence in bronectin-coated wells for 24-36 hours in DMEM supplemented with 1% FBS.Cell monolayers were wounded with sterile pipette tips and washed twice with medium, prior to treatment with 10mg/ml CAR or mCAR peptides or vehicle control in the presence of DMEM supplemented with 1% FBS.Time-lapse bright eld images were acquired on a 3i Marianas live-cell imaging system using a Zeiss 10x/NA0.45Plan-Apochromat objective or Zeiss Apotome2 wide eld microscope using a Zeiss 10x/NA0.3M27 EC Plan-Neo uar objective.Point visiting was used to allow multiple positions to be imaged within the same timecourse and cells were maintained at 37°C and 5% CO 2 .Images were collected every 10 minutes for up to 21 hours using a Hamamatsu ORCA-Flash4.0v2 sCMOS camera on the 3i Marianas or a Zeiss Axiocam 506 mono on the Zeiss Apotome2.Percentage scratch wound closure was calculated by using ImageJ to measure the area of the gap between leading edges of each cell monolayer at the beginning and end of the time-lapse movie.Individual cell migration was tracked manually using the MTrackJ plugin for ImageJ.Migration tracks were plotted using the Chemotaxis Tool and Manual Tracking ImageJ plugins.
siRNA-Mediated Knockdown Transfections siRNA-mediated knockdown was achieved by using an Amaxa Nucleofector IIb system according to manufacturer's instructions.Brie y, TIF or HaCaT cells were cultured to 70% density before trypsinisation and approximately 2.5X10 6 cells were used per reaction.TIF cells were transfected using Nucleofector Kit NHDF, program A-023 and 300 nM oligonucleotides.HaCaT cells were transfected using Nucleofector Kit V, program U-020 and 300 nM oligonucleotides.Cells were transfected with 300 nM human SDC4 siRNA (Ambion Silencer Select Oligo #1 s12638 or Oligo #2 s12639), human ARF6 siRNA (Dharmacon, sequence (sense) 5′-CGGCAUUACUACACUGGGA-3′), or AllStars Negative Control siRNA (Qiagen).The cells were subject to two rounds of knockdown transfection with 48-hour intervals.Experiments were performed 48 hours after the second transfection.Levels of protein knockdown were assessed by ow cytometry or immunoblotting.

Single-cell RNA-Seq
An extensive single-cell RNA-Seq (scRNA-Seq) analysis of cutaneous wound healing in mouse by Haensel et al. was taken for analysis 21 .The experiments include scRNA-Seq data of three wounded mouse skin samples, with a total of 16,428 cells and 27,998 genes cataloged, which were taken for further analyses.
Subsequent analyses were performed with the SCANPY Python library 66 , where default settings were used unless otherwise stated.Cells were ltered based on read counts (800 < x < 45,000) and number of genes expressed (x > 300), and genes were ltered based on number of cells expressed in (x > 0.5%), resulting in a ltered set of 16,351 cells and 13,807 genes.Counts were log-transformed (SCANPY function 'pp.log1p'), batch correction was performed using the Python implementation (Pedersen 2012) of the ComBat algorithm 67 ('pp.combat')based on the three samples as independent batches.Highly variable genes (HVGs) were determined ('pl.highly_variabl_genes') using the 'seurat_v3' avor and the 'counts' layer, with the top 6,000 HVGs kept for subsequent analyses.Principal component analysis ('pp.pca') was performed using the HVGs and 40 components, and nearest neighbors ('pp.neighbors')calculated with local neighborhood size of 15.The Python implementation (Traag 2017) of Louvain clustering 68 was used to cluster cells at r = 0.75 resolution.Uniform manifold approximation and projection (UMAP; 'tl.umap') modeling was used to visualize the clusters in 2D space.Ranked lists of genes and corresponding log-fold changes were generated for all cell clusters identi ed by Louvain clustering ('tl.rank_genes_groups'), which were subsequently used to assign cell type annotations based on review of existing literature.Cells which were identi ed as satellite cells or skeletal muscle were removed from the nal analysis and gures.

Statistical Analysis
The distribution of all in vivo data was determined by various normality tests (Shapiro-Wilk, Anderson-Darling, robust Jaque-Bera).Homogeneity of variances across groups was checked by Levene's and Bartlett tests.As our data deviated from Gaussian normal distribution, non-parametric tests were applied.
For comparison of two groups, Wilcoxon-Mann-Whitney test (with tie correction) was used.For comparison of multiple (> 2) groups, Kruskal-Wallis rank sum test with post-hoc Dunn's test for pairwise comparisons of independent samples was used.For categorical data, Pearson´s chi-square test with post-hoc Fisher exact test was employed.The p-values were adjusted for multiple comparisons by the Holm method.
Funding acquisition: MH, ER, MM, TJ Project administration: ER, MM, TJ Supervision: ER, MM, TJ Writing -original draft: BS, UM, ER, MM, TJ Writing -review & editing: All authors COMPETING INTERESTS E.R. has ownership interest (including patents) in Vascular Biosciences Inc., biotech company developing the CAR peptide for clinical applications.No potential con icts of interest were disclosed by the other authors.DATA AND MATERIALS AVAILABILITY data are available in the main text or the supplementary material.Crown Street, Liverpool, L69 3BX, UK Phone: +358-44-285 4620 (Cell) +44-151-795-4992 .Blondel, V. D., Guillaume, J. L., Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks.Journal of Statistical Mechanics: Theory and Experiment 2008, (2008).

Figures Figure 1 CAR
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Figure 2 CAR
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Figure 3 CAR
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Figure 7 CAR
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